Summary Heart failure manifests as impaired activation or deactivation of the heart at the level of the myofilament-the protein assembly that is responsible for generation of Ca2+-regulated contractile force. Nearly 6 million Americans suffer from congestive heart failure. 1 in 500 Americans have an inherited cardiomyopathy that is caused by a mutation in a myofilament protein. Due to its clinical significance, the cardiac myofilament is the subject of intense investigation; studies are aimed at defining its operation, defining its role in the pathophysiology of heart disease, and identifying promising strategies for treating heart disease. Cooperative activation-A central question in the field of myofilament regulation is how the various components of the myofilament communicate allosterically at the kinetic, energetic and structural level to rapidly switch on and off contractility. Of particular interest is how the myosin motors themselves contribute to their own regulation, a phenomenon called activation- dependent regulation or simply cooperative activation. Cooperative activation is dramatically illustrated in force vs. Ca2+ (force-pCa) measurements of permeabilized cardiac fibers, where Hill coefficients of 4 or more are routinely observed. Ca2+ sensitivity-The contractility and periodicity of the heart is regulated on a beet-to-beet basis to meet the needs of demands of the body for perfused blood. Mechanical and chemical signals such as cell stretch, neurohormonal and redox regulatory pathways manipulate myofilament activation to tune cardiac output. In perfused whole hearts, these manipulations alter the time-dependent changes in intraventricular pressure and volume. These manipulations cause altered myofilament sensitivity to Ca2+ in force-pCa measurements. Inherited cardiomyopathies are characterized in terms of their effect on the sensitivity of the myofilament to Ca2+. Cooperative activation and Ca2+-sensitivity are the two most prominent physiologic features of myofilament regulation. Despite decades of research their mechanisms remain unknown. The goal of this project is to establish mechanism of cooperative myofilament activation (Aim 1) and to establish the mechanism of altered myofilament sensitivity to Ca2+ (Aim II).